Formation of adjuvant complexes

ABSTRACT

&#34;Iscom&#34; adjuvant matrices, comprising a sterol, a glycoside, a solubilized water-insoluble antigen and, optionally, a phospholipid, may be formed without removing the solubilizing agent used for the antigen. The glycoside is preferably Quil A and the sterol is preferably cholesterol.

The present invention relates to processes for the formation ofcomplexes having adjuvant activity, the complexes comprising lipid andat least one glycoside.

Prophylactic immunisation of animals against microbes comprisesadministration of an antigen derived from the microbe in conjunctionwith a material that increases the antibody and/or cell-mediated immuneresponse of the antigen in the animal. This material is known as anadjuvant. The only adjuvants currently authorised for use in humans orpigs in many countries are aluminium hydroxide and aluminium phosphate.Although these adjuvants are sufficient for many vaccines, studies haveshown that Freund's complete adjuvant (FCA) or Quil A (also known assaponin) are often more efficacious in eliciting antibody response andcell mediated immunity but unfortunately these adjuvants may cause theanimals to react adversely to vaccination.

No. EP-A-0 109 942 and No. EP-A-0 180 564 describe immunogenic complexesformed between glycosides, such as triterpenoid saponins (particularlyQuil A), and antigens which contain a hydrophobic region. Theseimmuno-stimulating complexes have been given the name "iscoms". Theamount of Quil A in an iscom can be about 10 to 100 times lower thanwhen Quil A is mixed with the antigen to produce the same antigeniceffect. Iscoms do not create the adverse reactions associated with QuilA.

No. EP-A-231 039 indicates that the presence of antigen is not necessaryfor formation of the basic iscom structure, hereafter referred to as theiscom "matrix", it being possible to form the matrix from a sterol, suchas cholesterol, a phospholipid, such as phosphatidylethanolamine, and aglycoside such as Quil A.

In all three of the cited prior art documents, when a water-insolubleantigen was used, this was solubilised with a detergent and then, aftermixing with the glycoside etc, the detergent was removed, for example bydialysis, to cause formation of the iscoms. We have now found thatiscoms will form in the presence of detergent and that this removal stepis unnecessary.

One aspect of the invention provides a process for making an immunogeniccomplex comprising a water-insoluble antigen, the process comprisingsolubilising the antigen with a solubilising agent, admixing thesolubilised antigen, a glycoside, a sterol and, optionally, aphospholipid and forming an iscom substantially without removal of thesolubilising agent.

The invention also encompasses a vaccine prepared by such a process forhuman or animal use.

By "animal", we mean non-human vertebrate, preferably a mammal such as acow, pig, sheep, goat, horse, dog or cat.

The vaccine may comprise one or more diluents and carriers of aconventional nature.

By "antigen" we mean any entity capable of producing a protectiveantibody or cell-mediated immunological response against a pathogenicorganism in a vertebrate exposed to the antigen. The antigen may be allor part of a protein, glycoprotein, glycolipid, polysaccharide orlipopolysaccharide which is associated with the organism, or it may be apolypeptide or other entity which mimics all or part of such a protein,glycoprotein, glycolipid, polysaccharide or lipopolysaccharide. Becausethe antigen may or may not be incorporated in the iscom matrix duringformation of the latter, If it is not incorporated, it is not necessaryfor it to have a hydrophobic region or to be linked to a hydrophobicgroup, as in prior art procedures. The organism itself need not bepresent in the vaccine but it can reduce the cost of the vaccine ifwhole cells, for example of Haemophilus pleuropneumoniae, are usedinstead of extracts. Pathogenic organisms include viruses, bacteria,mycoplasmas, fungi, protozoa and other parasites. By "mycoplasma", weinclude the closely related organisms known as ureaplasmas andacholeplasmas.

Bacteria of medical or veterinary interest include Mycobacterium (e.g.M. tuberculosis), Clostridium (e.g. C. welchii), Rickettsia,Spirochaetes (e.g. T. pallidum), Escherichia (e.g. E. coli),Staphylococci (e.g. S. aureus), Haemophilus (e.g. H. influenzae and H.Pleuropneumoniae, Bordetella, (e.g. B. pertussis), Vibrio (e.g. V.cholerae), Salmonella (e.g. S. typhi and S. paratyphi) Streptococci(e.g. S. aqalatiae), Neisseria (e.g. N. gonorrhoea), Pasteurella (e.g.P. multocida), Legionella (e.g. L. pneumoniae), Pseudomonas (e.g. P.malliae), Actinobacillus (e.g. A. pleuropneumoniae), Campylobacter (e.g.C. jejuni) and Listeria (e.g. L. monocytogenes). Particular antigensinclude the adherence factor in Coli, e.g. pili K 88, porin protein ine.g. Salmonella and outer membrane proteins from - B. pertussis andNeisseria meningitidis.

Vaccination of pigs against H. pleuropneumoniae is particularlypreferred.

Mycoplasmas of veterinary or medical interest include Mycoplasma bovis,M. gallisepticum, M. agalactiae, M. hyopneumoniae, M. pneumoniae, M.synoviae, M. arthritidis, M. capricolium, M. dispar, M. hominis, M.mycodiessubs capri, M. orale, M. oripneumoniae, M. pulmonis, M. cynos,M. hyorhinis, M. mycoides, M. salvarium and M. fermentans.

Fungi associated with disease include: Candida spp., Cryptococcus spp.,Aspergillus spp., Microsporum spp., Trichophyton spp. Epidermophytonspp. and Dermatophilus spp.

Parasites associated with disease include: Toxoplasma spp. such as T.gondii, Plasmodium spp. such as Plasmodium vivax, P. malariae, P.falciparum and P. ovale, Teileria spp. such as T. parvum, Eimeria spp.such as E. tenella, Entamoeba histolytica, anaplasma of various types,trematodes such as Schistosoma Haematobium, S. mansoni, and S.japonicum, trypanosomes, such as Trypanosoma gambiense, T. brusei, T.congolesi and hepatica fasciola, nematodes, such as Ascaris, Wuchereria,Toxocara canis and Onchocerca spp., and cestodes, such as Taenia spp.,especially T. ovis.

Viruses associated with disease include: Orthomyxoviridae such asinfluenza A,B,C, Paramyxoviridae, especially measles virus, mumps virus,parainfluenza 1,2,3, and 4 viruses, canine distemper virus andrinderpest virus, Rhabdoviridae, especially rabies virus, Retroviridae,especially feline leukaemia virus and bovine leukaemia virus,Herpesviridae, especially Pseudorabies, Coronaviridae (esp. bovine),Togaviridae, such as EEE, WEE, VEE (eastern, western and Venezuelaequine encephalitis), yellow fever virus, bovine virus diarrhoea virus,equine herpes virus and European swine fever virus, Parvoviridae (esp.FMDV), Adenoviridae (esp. canine), Papovaviridae, Reoviridae (e.g.rotavirus), Caliciviridae, Rhinoviridae, Aphthoviridae, Flaviviridae,Pestiviridae, Morbilliviridae, Orbiviridae, Arenaviridae, Poxviridae,Bunyaviridae, Iridioviridae, especially African swine fever virus and,among unclassified viruses, human hepatitis β-virus and Marburg/Ebolavirus. Many of these have envelopes from which suitable antigens can beextracted.

If the antigen is not incorporated in the iscom matrix as part of theiscom structure itself, it may be present on the outside of the iscommatrix or it may subsequently be integrated into the iscom matrix.Alternatively, it may be present entirely separately in solution ordispersion. Such processes and compositions are disclosed in U.S. Ser.No. 07/426,589, filed concurrently herewith and entitled "AdjuvantComplexes", the entire contents of which are incorporated herein byreference.

The iscom matrix may be prepared in the way disclosed in No. EP-A-0 231039, namely by mixing together solubilised sterol, glycoside and(optionally) phospholipid but with the removal of the solubilising agentbeing omitted. An electron micrograph of iscoms obtained from Quil A,cholesterol and phosphatidylethanolamine is depicted in FIG. 1 of No.EP-A-0 231 039 (magnification 116.000 x). If phospholipids are not used,two dimensional structures are formed. An example of such structures isdepicted in FIG. 2 of No. EP-A-0 231 039. This figure is an electronmicrograph (magnification 146.000 x) of structures obtained from Quil Aand cholesterol. The term "iscom matrix" is used to refer to both the3-dimensional and 2-dimensional structures.

The glycosides to be used in the process according to the invention maybe the same as those mentioned in No. EP-A 0 109 942 and No. EP-A-0 231039, the entire contents of which are incorporated herein by reference.Generally the glycosides are glycosides showing amphipathic propertiesand comprise hydrophobic and hydrophilic regions in the molecule.Preferably saponins are used, especially the saponin extract fromQuillaja saponaria Molina in the first place DQ-extract preparedaccording to K. Dalsgard: Saponin Adjuvants. Bull. Off. Int.. Epiz. 77and 7-8) 1289-1295 (1972) and Quil A, also prepared according to K.Dalsgaard: Saponin Adjuvants III Archiv fur die gesamte Virusforschung44, 243-254 (1974). Other preferred saponins are aescine from Aesculushippocastanum (T Patt and W Winkler: Das therapeutisch wirksame Prinzipder Rosskaatanie (Aesculus hippocastanum) Arzneimittelforschung 10 (4)273-275 (1960) and sapoalbin from Gypsophilla struthium (R Vochten. P.Joos and R Ruyssen: Physicochemical properties of sapoalbin and theirrelation to the foam stability. J. Pharm. Belg. 42 213-226 (1968)). Theuse of Quil A is especially preferred.

In the process according to the invention the glycosides are used in atleast the critical micelle-forming concentration. In the case of Quil Athis concentration is about 0.03% by wt. Generally, the molar ratio ofglycoside (especially when it is Quil A) to sterol (especially when itis cholesterol) to phospholipid is 1:1:0-1, ±20% (preferably no morethan ±10%) for each figure. This is equivalent to a weight ratio ofabout 5:1 for the Quil A: cholesterol.

The sterols used in the process according to the invention may be knownsterols of animal or vegetable origin, such as cholesterol, lanosterol,lumisterol, stigmasterol and sitosterol. Preferably, cholesterol is thesterol used in the process according to the invention.

It is preferred for a phospholipid to be used, so that 3-dimensionaliscoms are formed. Suitable phospholipids include phosphatidylcholineand phosphatidylethanolamine.

The solubilising agent may be any of those mentioned in No. EP-A-0 109942 or No. EP-A-0 231 039, for example a detergent, urea or guanidine.

Generally, a non-ionic, ionic or zwitter-ionic detergent or a cholicacid based detergent, such as sodium desoxycholate, can be used for thispurpose. Preferably, the detergent used is octylglucoside, nonylN-methyl glucamide or decanoyl N-methyl glucamide but alkylphenylpolyoxyethylene ethers are also suitable, especially a polyethyleneglycol p-isooctyl-phenylether having 9 to 10 oxyethylene groups which iscommercialized under the trade name Triton X-100R.

Because, in the process of the invention, the solubilising agent neednot be removed for formation of the iscoms, there is no need for anyultrafiltration, dialysis, ultracentrifugation or chromatographic stepin order for the iscoms to be formed.

The process according to the invention may be used for the preparationof immunogenic complexes from antigenic proteins or peptides which showamphipathic properties. These proteins or peptides may be membraneproteins or membrane peptides isolated from viruses, bacteria,mycoplasmas, protozoa, helminths or other parasites or animal cells. Itis known that the serine and threonine radicals present in thehydrophobic region (the membrane domain) of some viral membrane proteinsmay be esterified. Non-membrane proteins and non-membrane peptideswithout the desirable hydrophobic properties may be incorporated intothe immunogenic complexes after coupling these with peptides consistingof hydrophobic amino acids, with fatty acid radicals, with alkylradicals and the like. Viruses which do not have an envelope but whichcan yield antigens for coupling to hydrophobic groups includePicornaviridae such as foot-and-mouth disease virus, polio virus,Adenoviridae, Parvoviridae, such as feline pest virus and swineparvovirus, and Reoviridae, such as rotavirus. The proteins or peptidesmay also be prepared synthetically or by means of recombinant DNAtechniques. Generally, ultracentrifugation or dialysis is not sufficientfor the purification of an antigenic protein or peptide of naturalorigin. Preferably, the antigens are purified by means ofchromatography, e.g. on a column of DEAE-Sephadex or by means of immunoaffinity chromatography.

Suitable methods for isolation of antigens and linking to hydrophobicgroups are described in No. EP-A-109 942.

The dissolved or solubilized antigen is generally contacted with asolution containing the glycoside in at least the criticalmicelle-forming concentration, a sterol, and optionally a phospholipid.

If desired, the solutions of the immunogenic complexes obtained may belyophilized. The lyophilized preparations may then be reconstitutedbefore use by addition of water.

Further, the invention relates to a pharmaceutical compositioncontaining immunogenic complexes prepared by means of the first aspectof the invention. These properties may be obtained by preparing theimmunogenic complexes in a form suitable for parenteral administration,preferably injectable administration and, in particular, for injectionby the sub-cutaneous or intra-muscular routes. Generally, thepharmaceutical compositions contain the immunogenic complexes in anaqueous, physiologically acceptable medium, which, if desired, containsa buffer and/or a salt such as sodium chloride for adaptation of theosmotic pressure. These procedures are all well known in the art.

The following non-limiting examples illustrate various preferred aspectsof the invention.

EXAMPLES 1-6: Empty Iscom Matrix Preparation Reagents Required

Phosphate Buffered Saline (Dulbecco A, Oxoid)

Lipid Mix (see below)

10% w/v solution of Quil A (Superfos) in sterile distilled water.

Preparation of Lipid Mix (10 ml) Materials

(1) Chloroform (analar)

(2) Cholesterol (Grade I, from porcine liver, Sigma)

(3) Lα phosphatidyl choline (Sigma)

(4) Mega 9 or 10 (Sigma) or Triton. (Mega 9 is nonyl N-methyl glucamide,whereas Mega 10 is decanoyl N-methyl glucamide).

(5) Sterile distilled water

Method

(a) Prepare 20% w/v of Mega 10 in sterile distilled water: i.e. weighout 2 g Mega 10 and dissolve in 10 ml of water.

(b) Weigh 50 mg cholesterol and add to 50 mg of phosphatidyl choline.

(c) When the cholesterol has dissolved in the phosphatidyl choline add 2ml of chloroform.

(d) Add the 10 ml of 20% w/v Mega 10. The mixture will be milky-white inappearance. This will raise the volume to 12 ml. However, the chloroformwill evaporate upon stirring and the final 10 ml volume will beachieved.

(e) Stand on a magnetic stirrer in the hot room (37° C.), with the lidof the container removed. Upon continued stirring, the mixture willbecome more mobile and clear.

(f) Store at room temperature.

EXAMPLE 1

(1) Place 20 ml of PBSA into a sterile universal flask.

(2) Add 100 μl of a 10% solution of Quil A.

(3) Add 400 μl of the lipid mix.

(4) Whirlymix the solution at full speed for 10 seconds.

(5) Sterilise solution via passing through a 0.2 μm filter (Millipore).

(6) Decant small volume for Electron microscopy studies.

(7) Store at +4° C.

EXAMPLE 2

The method of Example 1 was followed, but with the following variants.

(1) Place 20 ml of PBSA into a sterile universal flask.

(2) Add 200 μl of a 10% solution of Quil A.

(3) Add 800 μl of the lipid mix.

(4) Whirlymix the solution at full speed for 10 seconds.

(5) Sterilise through a 0.2 μm filter.

(6) Decant a small volume for electron microscopy studies.

(7) Store at +4° C.

EXAMPLE 3

(1) 20 ml PBSA was added to an Amicon stirred cell with a 30K Mwtcut-off membrane.

(2) To this was added 800 μl of the lipid mix plus 200 μl of Quil A (10%solution).

(3) The solution was concentrated to 5 ml and 100 ml PBSA added.

(4) The above step was repeated x2.

(5) The 20 ml contents were filtered through a 0.2μ filter labelled andstored at +4° C.

EXAMPLE 4

(1) 20 ml PBSA was added to an Amicon stirred cell with a 30K Mwtcut-off membrane in place.

(2) 400 μl of the lipid mix plus 100 μl of a 10% Quil A solution wasadded.

(3) The above solution was concentrated to 5 ml and resuspended to 100ml with PBSA.

(4) The above step was repeated x2.

(5) The final 20 ml contents of the cell were then filter sterilisedthrough a 0.2 μm filter, labelled and stored at +4° C.

EXAMPLE 5

(1) 20 ml PBSA was added to an Amicon stirred cell, a 30K Mwt cut-offmembrane being in place.

(2) 200 μl of the lipid mix plus 50 μl of a 10% Quil A solution wasadded.

(3) The above solution was concentrated to 5 ml and resuspended in 100ml PBSA.

(4) The above step was repeated x2.

(5) The final 20 ml prep of the cell was then filtered through a 0.2 μmfilter, labelled and stored at +4° C.

EXAMPLE 6

(1) 20 ml PBSA was added to an Amicon stirred cell (30K Mwt cut-offmembrane).

(2) 100 μl of the lipid mix plus 25 μl of a 10% Quil A solution wasadded.

(3) The above solution was concentrated to 5 ml and resuspended with 100ml PBSA.

(4) The above step was repeated x2.

(5) The final 20 ml contents of the cell were filter sterilised througha 0.2 μm filter, labelled and stored at +4° C.

In electron microscopy studies, these iscom matrices appeared to be lessnumerous than in the other examples.

EXAMPLE 7: Mouse Studies: Efficacy

Female mice of an inbred strain (Balb/C) and of the same age (6-8 weeks)were used. The mice were obtained from the barrier maintained (SPF) unitof Harlan-Olac (Bicester). The National Cell Type Collection strain ofMycoplasma hyopneumoniae was used throughout this study. The organismwas cultured in a modification of Friis medium. The stock cultures weremaintained at -70° C. Each suspension for vaccine incorporation wasprepared by inoculating 40 ml of broth with 4 ml of the stock culture atpH 7.4 and then incubating at 37° C. for 3 days or until the pH droppedto 6.8. By this time the organism had reached the log phase of growth.The cultures were inactivated using binary ethylene imine, concentratedusing tangential flow ultrafiltration, and washed with phosphatebuffered saline (PBS). The concentration of antigen was adjusted suchthat all vaccines contained 10 μg total protein per 0.1 ml mouse dose ofvaccine.

The iscom preparations of Examples 3 to 6 were used as follows.

Eight groups of six mice were immunised on two occasions, each mousereceiving 0.1 ml of the following preparations.

    ______________________________________                                        Group  Vaccinate                                                              ______________________________________                                        A      10 μg M. hyopneumoniae alone                                        B      10 μg M. hyopneumoniae adjuvanted with Freund's                            Complete                                                               C      10 μg M. hyopneumoniae with iscom matrix containing                        10 μg QA/mouse dose                                                 D      10 μg M. hyopneumoniae with iscom matrix containing                        5 μg QA/mouse dose                                                  E      10 μg M. hyopneumoniae with iscom matrix containing                        2.5 μg QA/mouse dose                                                F      10 μg M. hyopneumoniae with iscom matrix containing                        1.25 μg QA/mouse dose                                               G      0 μg M. hyopneumoniae with iscom matrix containing                         5 μQA/mouse dose                                                    H      10 μg M. hyopneumoniae + 10.sup.7 cfu H.                                   pleuropneumoniae with iscom matrix containing 5 μg                         QA/mouse dose                                                          ______________________________________                                         QA = Quil A                                                              

Groups of mice were pre-bled via the tail vein prior to primaryimmunisation and then fourteen days later. Twenty-eight days after theprimary immunisation the animals received a further vaccination and werebled 7 days later and finally 28 days post secondary. Serum wasseparated from the blood on the day of collection and stored at -70° C.for analysis by ELISA. Mice were observed following vaccination for anyadverse local and systemic reactions.

Results

The results of the experiments designed to assess the adjuvant effect ofQuil A in an iscom preparation where the antigen is external to thepreparation are shown below in Tables 1 and 2.

The results clearly demonstrate that the ISCOM preparation with thehighest protein: Quil A ratio of 10 μg: 10 μg/mouse dose produces thegreatest response to the vaccine, giving a response as good as if notbetter than the Freund's adjuvanted vaccine.

Groups of mice receiving iscom vaccines containing less than 10 μg ofQuil A per mouse dose performed no better, in producing a response tothe antigen, than the unadjuvanted antigen alone.

No vaccine reactions either local or systemic were noted in any micereceiving iscom vaccine or with antigen alone. In mice immunised withFreund's, small granulomas approximately 3 mm were noted at theimmunisation point but as with all the other mice no signs of systemicdisturbances were noted.

                  TABLE 1                                                         ______________________________________                                        Anti M. hyopneumoniae response in mice as measured                            by the INDIRECT ELISA                                                         Groups/        Pre                                                            Time           Vaccination                                                                             28DP2.sup.o                                          ______________________________________                                        A              0.023     0.184                                                B              0.019     0.126                                                C              0.019     0.203                                                D              0.019     0.043                                                E              0.019     0.083                                                F              0.032     0.029                                                G              0.042     0.052                                                H              0.040     0.150                                                ______________________________________                                         Standard Deviations omitted                                                   DP2.sup.o - days post secondary vaccination                              

EXAMPLE 8: Mouse Studies: Evaluation of Empty iscom matrix as anadjuvant for Haemophilus pleuropneumoniae vaccine in mice

Mice were immunised with vaccines containing whole inactivated H.pleuropneumoniae bacteria and iscom matrix formulated with varyinglevels of Quil A. Mice immunised twice with a vaccine containing 10 μgQuil A/mouse gave a greater antibody response than mice immunised withequivalent numbers of bacteria in Freund's adjuvant. Lower doses of QuilA had little apparent adjuvant effect. No adverse local or systemiceffects were observed following administration of any vaccine containingiscom matrix.

The NCTC reference strain of H. pleuropneumoniae serotype 3 (Strain1421) was grown to log phase growth in Yeast extract medium supplementedwith B AND. The culture was inactivated with 0.2% formalin and washedthree times with phosphate buffered saline (PBS) to yield a washed,inactivated whole cell antigen preparation. The antigen was standardisedby enumeration of bacteria using a Neubaumer chamber with modified Thomarulings and was stored in PBS at 4° C. until use.

Groups of mice, six per group, were immunised with H. pleuropneumoniaealone, H. pleuropneumoniae in FIA, or H. pleuropneumoniae together withiscom matrix containing different levels of Quil A, as detailed below. Afinal group received both H. pleuropheumoniae and M. hyopneumoniaeantigens together with iscom matrix to investigate possible interactionbetween the two antigens. Mice received two subcutaneous injections of0.1 ml of the appropriate vaccine on two occasions with a 3 weekinterval between imunisations.

    ______________________________________                                        Immunisation of mice                                                          Group  Vaccination                                                            ______________________________________                                        1      None                                                                   2      Hpl alone                                                              3      Hpl in FIA                                                             4      Hpl with iscom matrix containing 10 μg QA/per                              mouse dose of vaccine                                                  5      Hpl with iscom matrix containing 5 μg QA/per                               mouse dose of vaccine                                                  6      Hpl with iscom matrix containing 2.5 μg QA/per                             mouse dose of vaccine                                                  7      Hpl with iscom matrix containing 1.2 μg QA/per                             mouse dose of vaccine                                                  8      Hpl with iscom matrix containing 5 μg QA/per mouse                         dose of vaccine                                                               and 10 μg/per mouse dose Mycoplasma hyopneumoniae                          antigen.                                                               ______________________________________                                    

Following primary vaccination there was little antibody response in anygroup. Following secondary vaccination there was a rapid and pronouncedrise in antibody levels in 2 out of the 8 groups immunised. The greatestresponse was seen in mice immunised with bacteria together with iscommatrix containing 10 μg/mouse Quil A which had a mean peak OD of 1.24(±0.17) Units of Optical Density (UOD) when measured one week aftersecondary immunisation. Mice immunised with bacteria and FIA had asimilar but slightly smaller antibody response with a mean peak OD of1.11 (0.08) UOD on the same occasion. Mice immunised with bacteriaalone, bacteria with lower doses of Quil A, or bacteria together with M.hyopneumoniae antigen all had similar, lower, antibody responses withpeak values of between 0.43 and 0.63 UOD when measured one month aftersecondary immunisation.

                  TABLE 2                                                         ______________________________________                                        Anti - H. pleuropneumoniae response, as measured by                           indirect ELISA                                                                          Pre-vaccination                                                                          28 DP2                                                   ______________________________________                                        A           0.87         0.189                                                B           0.170        0.428                                                C           0.185        1.002                                                D           0.135        1.161                                                E           0.131        0.626                                                F           0.164        0.645                                                G           0.119        0.620                                                H           0.227        0.480                                                ______________________________________                                    

We claim:
 1. A process for making an immunogenic complex comprising awater-insoluble antigen, the process comprising solubilising the antigenwith a solubilising agent, admixing the solubilised antigen, a glycosideand a sterol and forming iscoms without removal of the solubilisingagent.
 2. A process according to claim 1 wherein the solubilising agentis sodium desoxycholate, octylglucoside, nonyl N-methyl glucamide,decanoyl N-methyl glucamide or a polyethylene glycolp-isooctyl-phenylether having 9 to 10 oxyethylene groups.
 3. A processaccording to claim 1 wherein a phospholipid is admixed with thesolubilzed antigen, glycoside and sterol.
 4. A process according toclaim 3 wherein the phospholipid is phosphatidylcholine.
 5. A processaccording to claim 1 wherein the glycoside is Quil A.
 6. A processaccording to claim 1 wherein the sterol is cholesterol.